Efficient synaptic transmission requires the enrichment and specific localization of receptors on the postsynaptic membranes apposed to the transmitter release sites. In the central nervous system (CNS), ionotropic glutamate receptors are the major excitatory neurotransmitter receptors and are divided into three broad classes, termed AMPA-, NMDA-, and kainate-type receptors, on the basis of molecular and pharmacological criteria. The predominant charge carrier during routine fast excitatory synaptic transmission is the AMPA-type receptor. Functional AMPA receptors are constructed from subunits termed glutamate receptors subunits 1–4 (GluR1–GluR4).
The correlation between various neurological diseases and the structural organization of the AMPA receptor has been the focus of many recent studies. Furthermore, the role of certain growth factors in the regulation of this receptor type has been postulated. For example, O'Brien et al (Neuron, 23, 193, 1999) disclosed that Narp (neural activity-regulated pentraxin) could induce clustering of AMPA receptors. The Narp polypeptide, also called neuronal pentraxin II (NP2) was originally cloned by Tsui et al (J. Neurosci. 16, 2463, 1996) as a novel immediate-early gene (IEG) induced by seizure in rat hippocampus. NARP has been independently identified as the guinea pig sperm acrosome protein p50/apexin (Noland, T. D. et al. (1994) J. Biol. Chem. 269, 32607; Reid, M. S., and Blobel, C. P. (1994) J Biol. Chem. 269, 32619). O'Brien further provided evidence that Narp may form multimers that subsequently act directly on the AMPA receptor, specifically the GluR1-3 subunits, inducing their clustering. Narp appears to derive from both pre- and post synaptic sources. Taken together, these data suggest that Narp may function to facilitate the formation of new excitatory synapses. Since Narp is an Immediate Early Gene (IEG) regulated by synaptic activity, its dynamic expression provides a novel mechanism for activity-dependent synaptogenesis and synaptic plasticity.
U.S. Pat. No. 5,762,552 discloses purified Narp polypeptide, including its amino acid sequence, while WO 97/39133 also provides the polynucleotide encoding the Narp polypeptide, the expression vector containing the above polynucleotides sequence, and a host cell transformed with this vector. Based on the fact that Narp is useful for induction of dendritic neurite outgrowth as well as promotion of neural migration, this patent discloses a method for treating a patient having neuronal disorders, utilizing administration of Narp to the patient.
EP 1,101,820A1 discloses the nucleic acid sequences encoding both human neuronal pentraxin receptor (NPR) and pentraxin I (NP1), and the application is directed to pentraxin I. It only briefly mentions pentraxin II, which is Narp.
WO 00/75661 provides a method for identifying compounds that affect the formation of AMPA receptors into aggregates. WO 00/75661 discloses methods for treating a patient having disorders associated with either an increase or a decrease in the function/expression of Narp, by administering to the patient agents that augment or inhibit Narp function/expression, respectively. WO 00/75661 discloses stimulation of NARP expression or activity for treatment of neuronal cell disorders including stroke or brain or spine cord injury damage including ischemic injury.
Ischemia of the Brain
Brain injury such as trauma and stroke are among the leading causes of mortality and disability in the western world.
Traumatic brain injury (TBI) is one of the most serious reasons for hospital admission and disability in modern society. Clinical experience, suggests that TBI may be classified into primary damage occurring immediately after injury, and secondary damage, which occurs during several days post injury. Current therapy of TBI is either surgical or else mainly symptomatic.
Cerebrovascular diseases occur predominately in the middle and late years of life. They cause approximately 200,000 deaths in the United States each year as well as considerable neurologic disability. The incidence of stroke increases with age and affects many elderly people, a rapidly growing segment of the population. These diseases cause either ischemia-infarction or intracranial hemorrhage.
Stroke is an acute neurologic injury occurring as a result of interrupted blood supply, resulting in an insult to the brain. Most cerebrovascular diseases present as the abrupt onset of focal neurologic deficit. The deficit may remain fixed, it may improve or progressively worsen, leading usually to irreversible neuronal damage at the core of the ischemic focus, whereas neuronal dysfunction in the penumbra may be treatable and or reversible. Prolonged periods of ischemia result in frank tissue necrosis. Cerebral edema follows and progresses over the subsequent 2 to 4 days. If the region of the infarction is large, the edema may produce considerable mass effect with all of its attendant consequences.
Neuroprotective drugs are being developed in an effort to rescue neurons in the penumbra from dying, though as yet none has been proven efficacious.
Damage to neuronal tissue can lead to severe disability and death. The extent of the damage is primarily affected by the location and extent of the injured tissue. Endogenous cascades activated in response to the acute insult play a role in the functional outcome. Efforts to minimize, limit and/or reverse the damage have the great potential of alleviating the clinical consequences.
Taipoxin
Taipoxin is a presynaptic toxin contained in the venom of the Australian taipan snake (Oxyuranus s. scutellatus). Fohlman J, (1976) Eur J Biochem 68 457–69. The intact complex molecule of taipoxin is composed of α, β and gamma (γ) subunits. Gamma-taipoxin is composed of 133 amino acids and has a molecular weight of 14.6 Kda. It is the only subunit of taipoxin which is N-glycosylated and sialyzed. Taipoxin is known to bind Narp; in fact, Narp was first purified on an affinity column of taipoxin (Kirkpatrick et al (2000) Biochemical Interactions of the Neuronal Pentraxins. The Journal of Biological Chemistry, 275, 23: 17786–17792), and identified through its interaction with taipoxin. In addition, it has been suggested that Narp and Narp receptor (NPR) participate in the internalization pathway of taipoxin into synapses (Dodds et al., Neuronal Pentraxin Receptor, a novel Putative Integral Membrane Pentraxin that Interacts with Neuronal Pentraxin I and II and Taipoxin-associated Calcium-binding Protein 49. The Journal of Biological Chemistry 272 (34) : 21488–21494, 1997) WO 01/63293 speaks of a screening method for agents effective for the treatment of schizophrenia, based, inter alia, on the susceptibility of cells exposed to Neural pentraxin I mediated activity to taipoxin. U.S. Pat. No. 4,341,762 concerns the possibility of using combinations of different types of toxins (among them taipoxin) isolated from snake venoms for treatment of neurological and related disorders. Of the 3 separate subunits of taipoxin, the αsubunit was found to be the most toxic (LD 50=300 ug/Kg—European Journal of Biochemistry (1979) 94, 531–540), while evidence of the toxicity of the γ subunit varies (from non-toxic to moderately toxic). The toxicity of the full taipoxin is 2 ug/Kg. The toxicity of α or α+β subunits is highly increased by addition of γ, suggesting that γ is involved in interaction with specific proteins on cell surface. The interaction possibly includes the carbohydrate moiety of γ subunit. The β subunit (β1 and β2) was found to be non-toxic and mitogenic (Lipps (2000) Toxicon 38 1845–1854), and has been proposed as a growth cell factor and for the treatment of wounds (U.S. Pat. Nos. 6,316,602 and 6,307,031).
U.S. Pat. No 6,316,602 relates to the use of beta-taipoxin as a cell-growth factor. This patent is directed primarily to methods of separating beta-taipoxin from the other subunits.
PCT publication No. WO 01/63293 is directed to identification of a long list of proteins and protein isoforms, and the use of these proteins and nucleic acids for screening, diagnosis and therapy of Schizophrenia. A screening method for treating schizophrenia which employs Pentraxin I in order to cause neuronal cells to be susceptible to taipoxin activity is disclosed.
None of the above publications teach or suggest inhibiting Narp in the context of ischemia, and certainly none of the above publications disclose beneficial effects of inhibiting Narp by the gamma subunit of taipoxin in connection with stroke, TBI or other ischemic conditions.